The ability to detect and/or quantitate the concentration of a pharmacological agent, metabolite, or toxin is a central aspect of modem diagnosis and management of disease. In some cases, such analytes can be detected directly by assaying their biological activities. In most cases, however, it is more efficient to detect such molecules by virtue of their capacity to specifically bind to antibodies, or by their physical characteristics, such as electrophoretic mobility.
Assays for the detection of target analytes involve the use of a binding partner which specifically binds to the analyte of interest in a sample to aid in the detection of the analyte. One type of assay for the detection of a target analytes are immunoassays. Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target analyte. The concept of immunoassays is based on a specific chemical reaction between an antibody and its corresponding antigen. Quantitation involves the separation of an antibody-bound antigen from the free antigen followed by the detection of an antibody-bound antigen or free antigen in solution, depending upon the specific analytical scheme. Such assays are used extensively in modem diagnostics (e.g., Fackrell, J., Clin. Immunoassay 8:213–219 (1985); Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, N.Y. (1985); Ngo, T. T., et al., In: Enzyme Mediated Immunoassay, Plenum Press, N.Y. (1985)).
There are many variations of assays for the detection of analytes, and the critical steps are either physical separation or discrimination and detection. Assays that require physical separation are termed “heterogeneous assays.” In contrast, homogeneous assays are designed such that the removal of bound from unbound species is unnecessary. Because homogeneous assays lack a separation step and are more easily automated, they are more desirable than heterogeneous assays in applications that entail the screening of large numbers of patients.
Analytes present at concentration levels below 10−9 M are generally assayed using either a competitive method or a direct method. In a competitive assay, the antigen of interest competes with a labeled antigen for a judicious amount of antibody. A direct assay is typically a sandwich assay involving two antibodies binding to different antigenic sites of an antigen. One antibody is bound to a solid phase material and is employed to harvest the antigen. The other antibody is labeled and used to generate quantitative information from the bound antigen (Cone, E. J., et al., J. Forens. Sci. 35:786–781 (1990); Baugh, L. D., et al., J. Forens. Sci. 36:79–85 (1991); Standefer, J. C., et al., Clin. Chem. 37:733–738 (1991)).
In order to facilitate the detection of binding between the analyte of interest and its binding partner, one or more reaction analytes is typically labeled (e.g., with a radioisotope, an enzyme, a fluorescent moiety, a chemiluminescent moiety, or a macroscopic label, such as a bead, etc.) (See Chard, T. et al., In: Laboratory Techniques and Biochemistry in Molecular Biology (Work, T. S., Ed.), North Holland Publishing Company, N.Y. (1978); Kemeny, D. M. et al. (Eds.), ELISA and Other Solid Phase Immunoassays, John Wiley & Sons, N.Y. (1988)). Radioisotopes have long been used in immunoassays. O'Leary, T. D. et al., for example, describe a radioimmunoassay for digoxin serum concentrations (O'Leary, T. D. et al., Clin. Chem. 25:332–334 (1979)). The difficulty of handling such hazardous materials and the problem of radioactive decay have led to the development of immunoassays that use other labels.
Fluorescent moieties are frequently used as labels in immunoassay formats (See Ichinose, N. et al., In: Fluorometric Analysis in Biomedical Chemistry, Vol. 10 (110), Chemical Analysis (Winefordner, J. D. et al., Eds.) John Wiley & Sons, N.Y. (1991)). For example, a fluorescence polarization immunoassay format for cocaine has been described (TDx®, Abbott Laboratories, Inc.) The TDx® format has also been used to assay acetaminophen serum levels (Koizumi, F. et al., Tohoku J. Exper. Med. 155:159-(1988); Edinboro, L. E. et al., Clin. Toxicol. 29:241-(1991); Okurodudu, A. O. et al., Clin. Chem. 38:1040 (1992)), and serum digoxin levels (Okarodudu, A. O. et al., Clin. Chem. 38:1040 (1992)). Wong, S. H. Y. et al., have described the use of an automated (OPUS) analyzer to measure the digoxin concentration in a monoclonal antibody mediated, fluorescence-based assay protocol (Wong, S. H. Y. et al., Clin. Chem. 38:996 (1992)). Lee, D. H. et al. also disclose the use of a fluorescence polarization assay and a chemiluminescent assay format to assay digoxin levels (Lee, D. H. et al., Clin. Chem. 36:1121 (1990)).
Electrophoretic methods have also been used to facilitate the detection of target analytes. Such methods exploit the fact that molecules in solution have an intrinsic electrical charge. Thus, in the presence of an electric field, each molecular species migrates with a characteristic “electrophoretic” mobility which is dependent upon the mass to charge the ratio of the molecular species. When this ratio is different from among the various species present, they separate from one another. Under the influence of such a field, all of the variants will move toward a designated charge opposite to the charge of the variants; those having a lower electrophoretic mobility will move slower than, and hence be separated from, those having a (relative) higher electrophoretic mobility.
Electrophoresis has been used for the separation and analysis of mixtures. Electrophoresis involves the migration and separation of molecules in an electric field based on differences in mobility. Various forms of electrophoresis are known, including free zone electrophoresis, gel electrophoresis, isoelectric focusing, and isotachophoresis. One approach to immunoassays employs capillary electrophoresis (CE) for the separation of free and bound label. In general, CE involves introducing a sample into a capillary tube, i.e., a tube having an internal diameter of from about 2 μm to about 2000 μm (preferably, less than about 50 μm; most preferably, about 25 μm or less) and applying an electric field to the tube (Chen, F-T. A., J. Chromatogr. 516:69–78 (1991); Chen, F-T. A., et al., J. Chromatogr. 15:1143–1161 (1992)). Since each of the sample constituents has its own individual electrophoretic mobility, those having greater mobility travel through the capillary tube faster than those with slower mobility. Hence, the constituents of the sample are resolved into discrete zones in the capillary tube during their migration through the tube. (Heegard, N. H. H., et al., Anal. Chem. 64:2479–2482 (1992); Gordon, M. J., et al., Anal. Chem. 63:69–72 (1991); F-T. A., U.S. Pat. No. 5,202,006; Chen, F-T. A., U.S. Pat. No. 5,120,413; Hsieh, Y-Z., et al., U.S. Pat. No. 5,145,567). The method is well suited to automation, since it provides convenient on-line injection, detection, and real-time data analysis. Capillary electrophoresis may be used to separate an antibody-antigen complex from either the unbound form of the antigen or the antibody. Either the bound or free species may be analyzed and quantitated. U.S. Pat. No. 5,863,401 to Chen discloses a method for the simultaneous quantification of multiple drug analytes in urine, based on combining immunochemical binding with capillary electrophoretic separation and laser-induced fluorescence.
Although sandwich-specific binding assays can provide a much higher sensitivity than competitive assays, the heterogeneity of labeled receptors and antibodies makes the capillary electroseparation difficult to carry out. This results because the unbound and complexed form of the receptor migrates non-uniformly, thus producing broad, poorly defined, rather than sharp, well-defined, distributions upon electroseparation analysis. Thus, conventional electroseparation methods may not offer significant advantages for specific binding assay applications.
Various approaches have been disclosed to overcome the inhomogeneity of large biomolecules. In one approach, the electrophoretic mobility of a labeled antibody is tailored by attaching charged groups to the same labeled molecule. In another approach, one antibody is labeled and the other is highly charged by means of a charge modifying moiety attached to the antibody.
U.S. Pat. No. 5,571,680 to Chen discloses a method of effecting the separation of an antigen or antibody from an antigen-antibody complex by CE by modulating the electrophoretic mobility of the antigen or antibody by chemical modification with well-defined charge-bearing organic molecules, such as oligonucleotides.
U.S. Pat. No. 6,103,537 to Ullman et al. discloses a method for masking inhomogeneity of an antibody in assay mixtures that are to be separated by CE. The method involves preparing labeled reagent particles comprising a labeled analyte-specific antibody bound to insoluble particles, and incubating the sample containing an analyte of interest with the particle-bound antibodies to form a complex between the analyte and the labeled particle-bound antibody. The reaction mixture is separated by CE, and the labeled complex is detected to determine the concentration of the analyte. However, it is well known that in the preparation of such labeled reagent particles, it is impossible to control the amount of antibody that becomes bound to the insoluble particle. As a result, different particles will contain different amounts of immobilized antibody relative to the other particles and will therefore bind to different amounts of analyte. Consequently, the various labeled complexes formed between the reagent particles and the analyte in the sample will naturally be inhomogeneous and the signal detected from the labeled complexes will be broad. Therefore, it will be more difficult to accurately correlate with the concentration of the analyte, as well as to cleanly separate the complexed label from the free label.
Reagent particles comprising latex particles coated with avidin (latex-avidin) are key intermediates in the manufacturing of latex-avidin-bidentate reagents for the Synchron CX®, Synchron LX®, and Immage® systems of Beckman Coulter (U.S. Pat. Nos. 5,747,352; 5,422,281; and 5,196,351). However, there exists no in-process testing of the latex-avidin intermediate, because reliable direct methods are not available. Further, currently available methods are only applicable for analyzing free avidin in solution rather than assessing the concentration of avidin that is coated on the latex particle. Without knowledge of the avidin loading on the latex particles and consistency with previous lots, a general practice is to simply proceed to the next and final step by coupling the latex-avidin intermediate with the bidentate reagent to produce the latex-avidin-bidentate reagent. Since the bidentate coupling step is a simple and reliable one, failure in manufacturing the latex-avidin-bidentate reagent is then attributed to the latex-avidin material. When failure occurs, the bidentate reagent is wasted, the latex-avidin material has to be disposed of, a large amount of washing buffer is consumed, and most importantly, time is wasted. A common method to qualify the in-process latex-avidin intermediate is to couple a small amount of the latex-avidin material to a small quantity of the bidentate material, followed by immunoreactivity assessment of the resulting conjugate reagent on Synchron CX® for its acceptability. This method, however, is labor intensive and time-consuming, requiring three to ten days to obtain the results. Thus, there is still a need for a simple, fast, and reliable method for the in-process testing of latex-avidin intermediates.
In view of the importance of accurately detecting and quantitating analytes in samples, it would be desirable to provide processes which combine the advantages of particle-enhanced assays, capillary electrophoresis, and fluorescent detection techniques to rapidly detect target analytes in a test sample. In particular, it would be desirable to provide processes for more accurately analyzing samples for analytes of interest using particle-enhanced assay procedures and capillary electrophoresis detection techniques. The present invention provides such methods.